ABSTRACT: During tidal emersion, microphytobenthic algae (principally pennate diatoms) migrate vertically within the top mm of sediment to form transient biofilms. This migration has been described as an evolutionary strategy, maximizing photosynthetic activity whilst minimizing photo-damage. This study investigated the effect of vertical migration on photophysiological parameters measured by pulse amplitude modulated (PAM) fluorescence, comparing migratory biofilms with artificially created non-migratory biofilms. Dark adaptation for 5 min, a light response curve and 18 min dark recovery period were sequentially applied to each biofilm treatment. Vertical migration and the variable chlorophyll fluorescence signal from cells migrating to the sub-surface significantly affected all variables measured (minimum fluorescence yield, F0, and maximum yields, Fm or Fm in the dark and light adapted states respectively). Maximum relative electron transport rates (rETRmax) calculated from light response curves were overestimated due to downward migration by cells in avoidance of high light. Changes in Fm due to vertical migration caused underestimation of non-photochemical quenching (NPQ). F0, often used as a proxy for algal biomass, was significantly lower on migrational biofilms after 5 min dark adaptation compared to non-migratory biofilms due to downward migration in the dark. This response to dark adaptation followed a temporal pattern, showing a stronger tendency for downward migration towards the end of the emersion period. Measurement of photophysiological variables of migrational microphytobenthic biofilms using variable chlorophyll fluorescence must take into account the effects of light induced vertical migration, in order to prevent errors in the calculation of derived parameters.